Sample preparation is a necessary step for the many genetic, biochemical, and biological analyses of biological and environmental samples. Sample preparation frequently requires the separation of sample components of interest from the remaining components of the sample. Such separations are often labor intensive and difficult to automate. In many cases, the preparation of a sample requires substantial purification of one or more components of the sample that can subsequently be analyzed. Dielectrophoresis, also sometimes called “conventional dielectrophoresis” to distinguish it from travelling wave dielectrophoresis, is the translocation of neutral or charged particles in response to an electric field of non-uniform magnitude. Dielectrophoresis and travelling wave dielectrophoresis provide efficient, reliable, nondisruptive, and automatable methods for the separation of particles in a sample based on their dielectric properties.
Dielectrophoresis and traveling-wave-dielectrophoresis have been used, for example, to separate breast cancer cells from blood (Becker et al., Proc. Natl. Acad. Sci. USA 92: 860-864 (1995)); to separate bacteria from blood cells (Hawkes et al., Microbios. 73: 81-86 (1993) and Cheng et al., Nat. Biotech. 16: 546-547 (1998)); to enrich CD34+ stem cells from blood (Stephens et al., Bone Marrow Transplantation 18: 777-782 (1996)); and to collect viral particles, submicron beads, and biomolecules (Washizu, et al, IEEE Trans. Ind. App. 30: 835-843 (1994), Green and Morgan, J. Phys. D: Appl. Phys. 30: L41-L44 (1997), Hughes et al., Biochim. Biophys. Acta 1425:119-126 (1998), and Morgan et al., Biophys. J. 77: 516-525 (1999). The separation of particles, including cells, using dielectrophoresis is also described in U.S. Pat. No. 4,390,403 issued Jun. 28,1983; U.S. Pat. No. 4,326,934 issued Apr. 27, 1982 to Pohl; U.S. Pat. No. 5,344,535 issued Sep. 6, 1994 to Betts and Hawkes; U. S. Pat. No. 5,454,472 issued Oct. 3, 1995 to Benecke et al.; U.S. Pat. No. 5,569,367 issued Oct. 29, 1996 to Betts et al.; U.S. Pat. No. 5,653,859 issued Aug. 5, 1997; U.S. Pat. No. 5,795,457 issued Aug. 18, 1998 to Pethig, et al.; U.S. Pat. No. 5,814,200 issued Sep. 29, 1998 to Pethig et al.; U.S. Pat. No. 5,858,192 issued Jan. 12, 1999 to Becker et al.; U.S. Pat. No.5,888,370 issued Mar. 30, 1999; U.S. Pat. No. 5,993,630 issued Nov. 30, 1999 to Becker et al.; U.S. Pat. No. 5,993,631 issued Nov. 30, 1999 to Parton et al.; and U.S. Pat. No. 5,993,632 issued Nov. 30, 1999 to Becker et al.
Dielectrophoresis refers to the translational movement of polarized particles in an alternating current (AC) electrical field of non-uniform magnitude. When a particle is placed in an electrical field, if the dielectric properties of the particle and its surrounding medium are different, dielectric polarization will occur to the particle. Thus, electrical charges are induced at the particle/medium interface. If the applied field is non-uniform, then the interaction between the non-uniform field and the induced polarization charges will produce net force acting on the particle to cause particle motion towards the region of strong or weak field intensity. The net force acting on the particle is called the dielectrophoretic force and the particle motion is dielectrophoresis. The dielectrophoretic force acting on a particle depends on the dielectric properties of the particle, the dielectric properties of the particle surrounding medium, the frequency and magnitude of the applied electrical field, and the field magnitude distribution. In the literature, dielectrophoresis is sometimes called conventional dielectrophoresis (e.g. in Wang et al. Biochim. Biophys. Acta 1243: 185-194 (1995)). For simplicity, in the present application, we use the term “dielectrophoresis” to refer the motion of polarized particles in a field of non-uniform magnitude. The corresponding forces that act on the polarized particles are “dielectrophoretic forces”.
Separation techniques that use dielectrophoresis are dielectrophoretic retention (or dielectrophoretic affinity), dielectrophoretic migration, and dielectrophoretic/gravitational-field flow fractionation (DEP/G-FFF). In dielectrophoretic retention, one or more particles of a sample that experiences a positive dielectrophoretic force is attracted to and retained in one or more areas of a chip or chamber that are regions of electric field maxima, typically at electrode edges. In separation procedures where dielectrophoretic retention is used, components of a sample that experience negative dielectrophoretic forces or weakly positive dielectrophoretic forces generally are flushed out of the chamber by fluid flow. In dielectrophoretic migration, particles are separated based on the different dielectrophoretic forces they experience. For example, one or more particles can experience negative dielectrophoretic forces and migrate to one area of a chamber and one or more different particles can experience positive dielectrophoretic forces and migrate to a different area of a chamber. In dielectrophoretic/gravitational-field flow fractionation (DEP/G-FFF), particles can be levitated by negative dielectrophoretic forces. Different particles are levitated to different extent in the chamber and are subjected to a fluid flow profile. Different particles levitated to different heights in the chamber are carried with the fluid flow at different speeds and thereby separated.
Travelling wave dielectrophoreis (TW-DEP), also called “travelling wave field migration” or TWFM, is related to dielectrophoresis (or sometimes called conventional dielectrophoresis), described above. In travelling-wave dielectrophoresis, the electric field has a non-uniform phase distribution. The travelling-wave electric field interacts with the field-induced polarization of particles and generates forces acting on the particles. Particles are caused to move either with or against the traveling direction of the traveling field. Travelling-wave dielectrophoretic forces depend on the dielectric properties of the particles and their suspending medium, on the frequency and magnitude of the traveling field, and on the phase value distribution of the traveling field. In “2-dimensional dielectrophoresis” (2-D DEP) particles are separated by exploiting both dielectrophoretic forces and travelling wave dielectrophoresis forces acting on particles (De Gasperis et al., Biomedical Microdevices 2: 41-49 (1999)). Furthermore, in 2-D DEP, particles are subjected to a fluid flow profile.
The theory for dieletrophoresis and traveling-wave dielectrophoresis and the use of dielectrophoresis for manipulation and processing of microparticles may be found in the following references: Wang et al. Biochim. Biophys. Acta 1243: 185-194.(1995); Wang et al. IEEE Transaction on Industry Applications 33: 660-669 (1997); Huang et al. J. Phys. D: Appl. Phys. 26: 1528-1535 (1993); Fuhr et al., Sensors and Materials 7: 131-146; Wang et al. Biophys. J. 72: 1887-1899 (1997); and Becker et al. Proc. Natl. Acad. Sci. 92: 860-864 (1995).
The manipulation of microparticles, including synthetic particles, crystals, colloids, molecules, compounds, molecular complexes, cells, and organelles, with dielectrophoresis and travelling-wave dielectrophoresis include concentration, aggregation, trapping, repulsion, linear or other directed motion, levitation, and separation of particles. Particles may be focused, enriched, and trapped in specific regions of the reaction chamber. Particles may be separated into different subpopulations over a microscopic scale, or may be transported over certain distances. The electrical field distribution necessary for specific particle manipulation depends on the dimension and geometry of electrode structures and may be designed using dielectrophoresis theory and electrical field simulation methods.
In many cases, however, it is difficult to separate components of a sample using dielectrophoretic forces and/or travelling wave dielectrophoretic forces because the dielectric properties of the moieties (e.g, cells or molecules) to be separated are similar. In other instances, it is difficult to separate moieties in a sample because of the small sizes of the moieties. Because the dielectrophoretic force on a moiety is proportional to the size of the moiety to be translocated, small moieties such as molecules (for example, nucleic acid molecules and proteins) require very large electric fields, and thus, very large applied voltages, for their manipulation. Such high voltages can damage biological materials, such as cells. High voltages can also cause heating of the separation medium, with potentially deleterious effects to both biological and nonbiological materials. There is a need for methods that can provide for the efficient separation of moieties regardless of their size or intrinsic dielectric properties.
Blood samples provide a special challenge for sample preparation and analysis. Blood samples are easily sampled from subjects, and can provide a wealth of metabolic, diagnostic, prognostic, and genetic information. However, the abundance of non-nucleated red blood cells, and their major component hemoglobin, can be an impediment to genetic, metabolic, and diagnostic tests. Removal of red blood cells can be achieved through centrifugation or filtering, which require extra steps and efforts that are not easily automatable. In other procedures, red blood cells are removed from blood samples by first lysing red blood cells and then collecting other cells through centrifugation. Lysis buffers used in these procedures tend to have undesirable effects on white blood cells. Thus there is typically a time window in which separation should be completed to avoid damage to the white blood cells, which can be inconvenient for the technician analyzing the sample. In addition, the use of centrifugation requires additional steps and efforts that are not easily automatable.